This invention relates to nucleic acid and amino acid sequences of two human insulin receptor tyrosine kinase substrates and to the use of these sequences in the diagnosis, prevention, and treatment of reproductive disorders, Alzheimer""s disease, cancer, immunological disorders, and disorders associated with insulin response.
Insulin controls blood glucose levels by stimulating glucose influx and metabolism in muscle and adipocytes and by inhibiting gluconeogenesis in the liver. Insulin also modifies the expression or the activity of a variety of enzymes and transport systems in nearly all cells.
Insulin action is mediated through the insulin receptor (IR), a transmembrane glycoprotein with protein tyrosine kinase (PTK) activity. Insulin binding triggers receptor autophosphorylation which activates PTK activity. The cellular response to insulin is mediated through tyrosine phosphorylation of cytosolic polypeptide substrates which act as second messengers in IR signal transduction. Once phosphorylated, the substrates bind to and activate various signal transduction proteins. The signal transduction proteins contain Src-homology-2 (SH2)-domains which bind phosphotyrosine-containing peptide motifs.
Several IR-PTK substrates have been described. The most extensively characterized substrate is the 185-kDa insulin receptor substrate-1 (IRS-1). IRS-1 is found in a variety of insulin responsive cells and tissues. It exhibits no intrinsic enzyme activity but, once phosphorylated, binds to and activates SH2-containing signal transduction proteins including phosphatidylinositol (PI) 3xe2x80x2-kinase and GRB-2, a regulator of the Ras pathway. (White, M. F. et al. (1994) J. Biol. Chem. 269:1-4.) Mutations in the IRS-1 gene impairs insulin-stimulated signaling and may contribute to insulin resistance in normal and diabetic populations. (Almind, K. et al. (1996) J. Clin. Invest. 97:2569-2575.)
Two 60-kDa protein substrates of the IR-PTK have been identified. One associates with the GTPase activator of Ras (termed GAP) and the other associates with PI 3xe2x80x2-kinase. (Yeh, T. et al. (1996) J. Biol. Chem. 271:2921-2928.) Two additional substrates for IR-PTK with molecular masses of 53 and 58 kDa were recently identified in rodents. These proteins, p53 and p58, are closely related and may arise from alternative splicing of mRNA or differential post-translational modifications. P53 and p58 do not associate with GAP or PI 3xe2x80x2-kinase and are immunologically distinct from the 60-kDa GAP-associated protein and the 60-kDa PI 3xe2x80x2-kinase-associated protein. P53 contains a microbodies C-terminal targeting signal which enables import of the protein into peroxisomes, glyoxysomes, and glycosomes. (Yeh, et al., supra.)
Post-receptor defects in the insulin signaling pathway are a common feature of type 2 (non-insulin-dependent) diabetes mellitus. (Stoffel M. et al. (1993) Diabetologia 36: 335-337.) Other disorders or conditions associated with disturbances in insulin response include hyperglycemia, myotonic muscular dystrophy, acanthosis nigricans, retinopathy, nephropathy, atherosclerotic coronary and peripheral arterial disease, and peripheral and autonomic neuropathies.
The discovery of two new human insulin receptor tyrosine kinase substrates and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of reproductive disorders, Alzheimer""s disease, cancer, immunological disorders, and disorders associated with insulin response.
The invention features substantially purified polypeptides comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 and SEQ ID NO:4 or a fragment of SEQ ID NO:2 and SEQ ID NO:4, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 and SEQ ID NO:4 or a fragment of SEQ ID NO:2 and SEQ ID NO:4. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 and SEQ ID NO:4 or a fragment of SEQ ID NO:2 and SEQ ID NO:4.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with insulin response, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention also provides a method for treating or preventing Alzheimer""s disease, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention also provides a method for treating or preventing an immunological disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
The invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3 in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.